The long-term objective of this research program is to provide a comprehensive understanding of the biochemical mechanisms and physiological regulation of intracellular protein degradation. Intracellular protein degradation plays a critical role in determining the net growth and atrophy of tissues, such as skeletal muscle. Moreover, qualitative changes in muscle protein composition that occur in response to altered patterns of contractile activity are brought about by regulated protein degradation. Mammalian cells contain multiple proteolytic systems for the degradation of their constituent proteins. The relative roles and contributions of these systems to the degradation of intracellular proteins under steady state conditions and under conditions of altered protein metabolism, such as occurred in skeletal muscles during exercise, are poorly understood and remain fundamental unresolved issues in muscle physiology. This research unit of the Program Project Grant will examine the relative roles of two major proteolytic systems, the ubiquitin-proteasome system and the calpain system, in the normal degradation of skeletal muscle proteins in cultured cells. Functions of each system and relative functions of specific components within each system will be examined after selective inhibition of expression of targeted components by RNA interference, and after overexpression of certain components by adenovirus infection. Effects of these treatments on rates of global and myofibrillar protein degradation, in vitro and in vivo protease activity, ubiquitination of cellular proteins, and the level and composition of various protease and protease-regulator complexes, will be determined. Finally, the effects of two models of altered contractile activity, chronic low-frequency stimulation of a motor nerve and extensive voluntary running, on the levels and function of the two proteolytic systems will be evaluated. These experiments will provide important new information about basic biochemical mechanisms of intracellular protein degradation in skeletal muscle, and define how the function and composition of proteolytic systems are regulated by specific patterns of contractile activity in a manner that promotes altered protein degradation.